螢光發報之原位適配接合反應應用於異戊二烯焦磷酸合成酶抑制劑的開發

張皓晴

doi:10.6342/NTU.2009.01738

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螢光發報之原位適配接合反應應用於異戊二烯焦磷酸合成酶抑制劑的開發

Fluorogenic In Situ Click Chemistry Applied to Inhbitor Discovery of Prenyl Pyrophosphate Synthases

張皓晴 , Masters　　Advisor：陳昭岑　　

繁體中文 DOI： 10.6342/NTU.2009.01738

螢光；原位適配接合化學；異戊二烯焦磷酸；抑制劑；篩選； fluorogenic ； in situ click ； prenyl pyrophosphate ； inhibitor ； screening

Abstract │ Reference (75) │ Altmetrics

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Isoprenoids are widespread in the biological systems. For examples, abnormal cellular signal transduction via protein prenylation with C20-GGPP is observed in some tumor cells, and the C55-UPP serves as a lipid carrier in the bacterial cell wall biosynthesis. Therefore, developing inhibitors for prenyl pyrophosphate synthases has great implications in the biomedical applications. In this thesis, we have developed an effective screening method based on the fluorogenic in situ click reactions to fish out inhibitors for a series of prenyl pyrophosphate synthases more effecitvely.
     We designed and synthesized several hydrophobic 3-azidocoumarins and hydrophilic arylalkynes, to complement the hydrophobic tunnel and the polar catalytic region of the target enzymes. Utilizing the strategy of in-situ target-guided synthesis, we are hoping that the enzymes select the proper fragments themselves and further promote the azide-alkyne cycloaddition between the hydrophobic fragments having 3-azidocoumarin and the hydrophilic ones with alkyne functionality. Once the cycloadduct formed, the fluorescence of azicocoumarin is no longer suppressed by the azido group and the resultant fluorescence enhancement is a good indication that the target enzyme promotes the click reaction itself.
     The binding ability of 10 hydrophobic fragments to the active sites of UPPs was evaluated by fluorescence competition experiments by first incubating the enzyme with MANT-GPP, which was proven to be the substrate and inhibitor for UPPs and could give fluorescence increase at 425 nm once bounded. The titration results revealed that the binding ability of the hydrophobic fragments could attributed to the length of substituted hydrocarbon tails. Hence, we only employed three different lengths of prenyl substituent (denoted as Pre-hAzC, Ger-hAzC, Far-hAzC) and three hydrophilic arylalkynes (denoted as pPP, mPP, mBP) with potent pyrophosphate or bisphosphonate groups were chosen for further in situ click experiments. These fragments (3 x 3) were arranged in a spatial combinatorial fashion and each combination was incubated with three target enzymes (UPPs, OPPs or hGGPPs) for 3 days, respectively. The fluorescence was then measured by the fluorescence plate reader for each combination. These results showed that the combination of Pre-hAzC/mBP incubated with hGGPPs gave the best fluorescence with a 4.5-fold fluorescence enhancement among 27 (3 x 3 x 3) possible combinations. To confirm and verify which is the isomer of Pre-hAzC/mBP generated by the in situ click reactions, the possible cycloadducts of Pre-hAzC and mBP, denoted as Pre-1,4-mBP and Pre-1,5-mBP, were synthesized and tested their biological activity. The inhibition results revealed that the former suppresses 84% of hGGPPs activity at 100 uM concentration, the latter only inhibits 15% of hGGPPs activity. Moreover, the molecular modeling results of docking are consistent with the biological inhibition data.

Reference ( 75 ) 〈TOP〉

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